Ceramide is a lipid-derived second messenger that suppresses vascular smooth muscle (VSM) cell mitogenesis in vitro and in vivo. Multiple laboratories, including our own, have identified protein kinase C zeta (PKCzeta) as a target of ceramide. We have published data demonstrating that ceramide-activated PKCzeta is necessary for VSM cell growth arrest. Two signaling pathways have been identified that contribute to the anti-mitogenic actions of ceramide. Ceramide directly activates PKCzeta, to 1.) induce SAPK signal complex formation;and 2.) inhibit AKT1 signaling cascades. Elucidation of the biophysical and biochemical mechanisms by which ceramide activates PKCzeta is the focus of this new proposal. It is now hypothesized that ceramide stimulates PKCzeta;activity within discrete lipid microdomains or rafts. We will test this hypothesis in the following Specific Aims. In Specific Aim 1, we will identify ceramide-enriched lipid rafts as the site at which PKCzeta, is phosphorylated and optimally activated by upstream kinases. In Specific Aim 2, we will characterize and validate ceramide-binding domains or clefts on PKCzeta. These studies will elucidate the biophysical and biochemical mechanisms by which inflammatory cytokine receptor-induced ceramide formation limits cellular proliferation in models of inflammatory vascular diseases. The long-term innovation of these studies is the clinical potential of direct delivery of ceramide analogues to the site of dysregulated VSM growth observed after angioplasty and stent placement. Additionally, this proposal holds the promise of defining lipidomimetics suitable as therapeutics or screening tools for further drug discovery.